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226 Enhanced HIV-1 Gag Expression and Immunogenicity by an AAV-ITR DNA Plasmid Vector Encoding the Antigen as a Lysosomal Membrane Protein Chimera
E. T. A. Marques, Jr.*, P. Chykhlikar, Y. Lu, J. S. Chen, I. C. Leao, J. T. August, and C. Chougnet
The Johns Hopkins Sch. of Med., Baltimore, MD, USA
Immune responses to HIV-1 Gag correlate with better outcomes in infected patients and immune-based approaches to enhance the anti-Gag responses are considered for clinical application. However, the expression of Gag by DNA vectors has been hampered by the requirement for coexpression of Rev. We designed a novel strategy to increase Gag expression by (1) using a DNA plasmid containing the adeno-associated virus (AAV) inverted terminal repeats (ITRs), which increases expression 4-fold, and (2) inserting the Gag gene into the lysosome-associated membrane protein (LAMP) sequences, which results in a further 50-fold increase in expression as well as targeting of Gag to the MHC class II compartment. The combined effect is a greater than 200-fold increase in expression over the wild-type Gag using a conventional vector (pcDNA3.1), and more than 15-fold compared with a Gag expression system in which the inhibitory RNA sequences were removed (GagDINS). Mice immunized with the AAV-ITR LAMP/Gag vaccine produced markedly greater anti-Gag IgG titers (>1:218,000; IgG1/2a ratio of 9) than those immunized with other plasmids and the Ab titer was maximal after 2 immunizations. Cytokine expression/production in response to p55-Gag protein indicated a strong CD4-mediated activation in mice vaccinated with AAV-ITR LAMP/Gag. IL4 and INFgamma mRNA expression were increased by 15- and 2-fold, respectively, compared with the Gag wild-type. Gag-specific INFgamma production induced by AAV-ITR LAMP/Gag vaccine was markedly greater (~10-fold) than that induced by the secreted form of the LAMP/Gag chimera, which expresses an equal amount of protein but does not target the class II compartment. The AAV-ITR LAMP/Gag DNA also induced strong CD8-mediated responses after recombinant vaccinia boost, as analysed ex vivo: 6.3% tetramer binding and 4.1% intra-cellular staining of total CD8+, and 55% cytotoxicity at 100:1 E:T ratio. These results indicate that our vaccine strategy induces a strong CD4-mediated response and good priming for CD8 responses against HIV Gag, which can be attributed to high Gag expression and LAMP trafficking to MHC II, and is a candidate for human application.
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